Imaging conformations of holo- and apo-transferrin on the single-molecule level by low-energy electron holography

Conformational changes play a key role in the biological function of many proteins, thereby sustaining a multitude of processes essential to life. Thus, the imaging of the conformational space of proteins exhibiting such conformational changes is of great interest. Low-energy electron holography (LEEH) in combination with native electrospray ion beam deposition (ES-IBD) has recently been demonstrated to be capable of exploring the conformational space of conformationally highly variable proteins on the single-molecule level. While the previously studied conformations were induced by changes in environment, it is of relevance to assess the performance of this imaging method when applied to protein conformations inherently tied to a function-related conformational change. We show that LEEH imaging can distinguish different conformations of transferrin, the major iron transport protein in many organisms, by resolving a nanometer-scale cleft in the structure of the iron-free molecule (apo-transferrin) resulting from the conformational change associated with the iron binding/release process. This, along with a statistical analysis of the data, which evidences a degree of flexibility of the molecules, indicates that LEEH is a viable technique for imaging function-related conformational changes in individual proteins.

www.nature.com/scientificreports/ clearest structural alteration, the conformational change associated with the transition between the holo-and apo-transferrin forms also affects the relative orientation of the lobes within the molecules via a readjustment of the connecting region between the two lobes 10,14 . Since transferrins have been extensively studied both experimentally and computationally [8][9][10][11][12][13][14] , these structural changes are well-understood and thus set a benchmark for assessing the performance of our LEEH investigation regarding the mapping of a protein's conformational space defined by a biologically relevant conformational change.
Here, by separately depositing holo-and apo-transferrin using native Electrospray Ion Beam Deposition (ES-IBD) 17 on a single layer graphene (SLG) substrate and subsequently investigating the observed conformational space with LEEH, we show that LEEH imaging can map the open and closed lobe conformations associated with the holo-and apo-forms of the molecules. Multiple molecular shapes are observed in the LEEH images of both types of molecules, consistent with different adsorption orientations with respect to the graphene surface. The resulting variety of views is particularly significant since not all possible orientations allow the identification of the exact lobe conformation (open or closed). While in the case of holo-transferrin, all the imaged molecules show lobes in a closed conformation, for apo-transferrin the different molecular orientations lead to a more complex scenario in which clear clefts are only visible on a subset of the molecules. Our statistical analysis shows that the amount of open lobe conformations observed in the LEEH experiments is in agreement with the percentage of clefts expected to be observable from a set of random orientations created from projections of the PDB model 15 of an apo-transferrin molecule. Furthermore, the evaluation of the observed molecular size distribution reveals that the sizes of the individual lobes match those expected from the models, while the overall length of the molecules is on average slightly longer than expected, which indicates a degree of flexibility of the connecting region. These  15 ) and holotransferrin (PDB: 1JNF 16 ). Transferrin molecules switch between the apo-and holo-forms by the binding and releasing of iron ions (indicated as red spheres in the holo-transferrin model). The associated conformational change manifests by the appearance of clefts in the lobes of apo-transferrin molecules.) b) One-step amplitude reconstruction of a hologram of an individual apo-transferrin molecule experimentally acquired by a LEEH measurement. On one of the lobes, the cleft defining the open lobe conformations is clearly visible, as indicated by the green arrow. Scale bar: 5 nm. (c) One-step amplitude reconstruction of a hologram of an individual holotransferrin molecule experimentally acquired by a LEEH measurement. Scale bar: 5 nm. Insets in (b) and (c) show the experimental image superimposed by a projection of the PDB model in an orientation corresponding to that of the imaged molecule.

Results
Separate samples of human apo-and holo-transferrin molecules were created by bringing the respective molecules from pure solutions into the gas phase via native electrospray ionization, mass-selecting to avoid contamination ( Supplementary Fig. 1), and subsequently soft-landing (landing energy < 5 eV per charge) the molecules on single layer graphene substrates at room temperature. The resulting apo-and holo-transferrin samples were imaged in our LEEH microscope (see Methods section), recording holograms of individual proteins. A one-step numerical reconstruction algorithm 2,3,18 (see Methods section) was employed to retrieve a real-space image from the experimentally acquired hologram, yielding a 2D projection of the molecular shape and size. For details on the above steps, see Methods section.
In general, the ES-IBD sample preparation process will generate proteins in a variety of different orientations with respect to the graphene surface 3 . In the case of transferrin, this results in a range of orientations from molecules showing both lobes ( Fig. 1b and c, approximately 40-65% of the total amount of observed molecules on a sample) to molecules exhibiting a compact structure with one of the lobes eclipsed by the other. The resulting diversity in orientation on the single layer graphene surface, and thus in overall molecular shape, can be mapped by LEEH imaging (see Supplementary Fig. 2). For each of the observed shapes, a matching projection from the crystallographic model can be found, indicating that both apo-and holo-transferrin molecules remain close to the native structure during ES-IBD sample preparation and LEEH imaging. Since the observation of molecules clearly exhibiting both lobes is beneficial for distingusihing open and closed lobe conformations, we initially focus our discussion on these structures. Fig. 1b and c show an example of an apo-and a holo-transferrin molecule with two clearly visible lobes, respectively. While the holo-transferrin molecule (Fig. 1c) presents the two lobes in a similar, compact shape corresponding to a closed lobe conformation, the apo-transferrin molecule exhibits a clearly distinguishable cleft in one lobe (Fig. 1b). This difference between holo-and apo-transferrin is validated by comparing a large number of reconstructed images obtained by LEEH imaging: lobes that are unequivocally associated with open conformations, i. e. lobes with a clearly visible cleft, are only observed in the samples prepared from apo-transferrin molecules. The clefts obtained from LEEH imaging have an average width at maximum opening of 2.1 ± 0.5 nm and an average length of 1.9 ± 0.5 nm , which matches the dimensions of the cleft observable in X-ray crystallography structures of apo-transferrin (e.g. 1RYX 15 ). Next to the experimental data, a projection of the crystallographic model (PDB: 1JNF 16 (holo), PDB: 1RYX 15 (apo)) in an orientation corresponding to that of the experimentally observed molecule is displayed in the insets of Fig. 1b and c. The reconstructed images closely resemble the projections of the model both in overall shape and size and on the level of the individual lobes for both molecules. While the overall molecular dimensions are almost identical in the case of apo-transferrin, a slight discrepancy in the molecule's length is apparent for holo-transferrin. This deviation does not affect the identification of open and closed lobe conformations and will be further discussed in the context of the statistical analysis presented in Fig. 3. It is relevant to note here that the projected model reproduces the cleft defining the open lobe conformation of the apo-transferrin molecule while simultaneously accounting for the bulky appearance of the other lobe, in which the cleft is not visible. Indeed, because of the clefts' relative locations, defined by the orientation of the two lobes with respect to one another 10 , only one cleft is visible while the cleft on the other lobe remains obscured. Hence, the projection of one of the lobes in an open conformation resembles that of a closed lobe. This similarity is evident both on the level of the crystallographic structures of apo-and holo-transferrin as well as when comparing the images reconstructed from experimental holograms, in which closed lobe conformations, as exhibited by holo-transferrin molecules, and eclipsed-cleft open lobe configurations in apo-transferrin molecules are virtually indistinguishable ( Fig. 1b and c).
Despite the fact that both lobes of an apo-transferrin molecule are in an open conformation, our statistical analysis of the experimental data shows that none of the observed molecules features two lobes clearly presenting a cleft. Additionally, lobes exhibiting a clearly recognizable open conformation were only observed in 18% of the imaged apo-transferrin molecules, where a high-contrast cleft is defined by a drop in amplitude values to background level, as in the example in Fig. 1b. When taking into account more ambiguous lobe conformations that fit an open lobe conformation in overall shape, but do not feature a high-contrast cleft, which could be due to a reduction in contrast due to a degree of fuzziness at the boundary of the molecules, the percentage of observable open conformations in apo-transferrin molecules increases up to 43% (Fig. 2a).
On the vast majority of the investigated molecules, our imaging does not observe cleft openings. Since the diversity in surface orientation effectively hinders the unequivocal determination of the lobe conformations on The comparison between experiment and model thus suggests a correspondence between the apo-transferrin X-ray structure (1RYX 15 ) and the on-surface apo-transferrin conformations measured by LEEH, confirming that the amount of experimentally observed clefts is reasonable for molecules whose structure closely resembles that of the crystallographic model. In addition, the observed molecular size distribution suggests a degree of flexibility of the connecting region in both apo-and holo-transferrin molecules. A statistical analysis of the lobe sizes (Fig. 3) shows that the size distribution of the individual lobes measured by LEEH matches the lobe sizes of the crystallographic model (long and short lobe dimensions of about 5.5 nm and 4 nm, respectively, as measured according to Fig. 3a and d). This confirms the preservation of the structural integrity of the molecules during the sample preparation process within the limit of the LEEH spatial resolution. When comparing the overall length of the molecules observed in extended two-lobe orientations to the full model length (maximum model length 9.5 nm for holo-transferrin and 10.5 nm for apo-transferrin), however, the experimentally measured structures (Fig. 3b and e) are on average slightly elongated, as demonstrated by the broad full-length distributions centred at 11.5 nm (holo) and 12.4 nm (apo), respectively ( Fig. 3c and f). This is further corroborated by comparing the size distribution of the individual lobes with the full-length distribution of the molecules (Table 1). For holo-transferrin, Gaussian fits of the lobe dimensions lead to peaks centred at 4.3 and 5.1 nm with standard deviation ( σ ) equal to 0.7 and 0.8 nm for short and long, respectively. A similar analysis on the full-length data leads to a Gaussian distribution centred at 11.5 nm with σ = 2.4 nm . In the case of apo-transferrin, the corresponding Gaussians are centred at 5.0 nm (short, σ = 0.8 nm ), 5.8 nm (long, σ = 1.1 nm ) and 12.4 nm (full length, σ = 2.5 nm ). Not only is the mean ( µ ) of the total molecular length larger than the sum of the means associated with the long lobe dimension for both holo-and apo-transferrin, but the standard deviation ( σ ) of the full-length distribution is larger than the standard deviation associated with a distribution generated by the sum of the distributions of the long dimension of the lobes ( σ Holo LongSum = 1.1 nm , σ Apo LongSum = 1.6 nm).
In summary, the statistical analysis of the molecular size distribution strongly points to a length variability of the connecting region between the two lobes for both apo-and holo-transferrin molecules, which appears to allow for an extension of the molecule while retaining the lobes' overall dimensions.

Conclusion
We have shown that low-energy electron holography imaging can map conformational differences intrinsically tied to the molecules' biological function which manifest on a spatial scale of approximately 1 nm on the single-molecule level as demonstrated by the structural differences observed in the reconstructed images of holo-and apo-transferrin. Additionally, the single-molecule nature of the LEEH measurement allows us to draw conclusions regarding the flexibility of the region connecting the two lobes from the analysis of the molecules'  Table 1. (d)-(f) Corresponding data for apo-transferrin. Table 1. Mean values and standard deviations of Gaussian fits of the size distributions in Figure 3 and comparison of the full molecule length distribution to the sum of two long lobe dimension distributions. Lobe dimensions fit well within the maximum and minimum dimensions that can be measured from the respective crystallographic models (maximum dimension holo = 6.5 nm and apo = 6.8 nm; minimum dimension holo = 4.1 nm and apo = 4.8 nm). The comparison of the experimentally measured full molecule length and the sum of the lobe dimensions indicates a broadening of the full molecule length distribution which is in agreement with the observed elongation of the experimentally observed molecules with respect to the crystallographic models, which yield the following projected full-length dimensions: 9.5 nm for holo-and 10.5 nm for apotransferrin. www.nature.com/scientificreports/ conformational space. Since many protein systems undergo conformational changes while fulfilling their biological role, the imaging of such structural differences on the single-molecule level could be of great relevance for the study of a large range of other proteins in a biological or biomedical context. Transferrin itself could be a system of interest for further investigation since it in principle can not only bind iron, but also other metal ions or small molecules 8,13,19,20 , which, along with its ability to cross the blood brain boundary, makes it a promising candidate for targeted drug delivery.

Methods
Low-energy electron holography. The LEEH microscope 3,4,21,22 used to obtain the experimental protein holograms features a lens-less in-line holography geometry 23,24 , i. e. the electron source, the sample and the detector are aligned along the same optical axis. Sharp tungsten tips terminating in a few atoms are used as coherent electron sources, which field-emit low-energy electrons (30-150 eV) when in measuring distance to the SLG substrate (approximately 100-500 nm), which allows the imaging of individual protein molecules. The magnification is purely geometrical and can be adjusted by a change in tip-sample distance. The hologram is formed as the interference pattern resulting from the superposition of the wave scattered by the proteins and the unscattered reference wave. The hologram is recorded as a photograph of the fluorescent screen of a microchannel plate detector and is reconstructed numerically via a wave field propagation-based algorithm 18,25 . The propagation can be described by a Fresnel-Kirchhoff diffraction integral of the form: where H(X, Y) is the measured hologram, R(X, Y) denotes the reference wave and The resulting wave field U(x, y) approximates the wave scattered by the object (exit wave). The absolute value of the exit wave yields the amplitude reconstructions presented in the main text, which as a first approximation can be viewed as a two-dimensional projection of the molecule along the optical axis. To simplify the numerical evaluation of the above integral, it can be rewritten as a series of Fourier transforms by employing the convolution theorem 18 . The LEEH microscope operates in an ultrahigh vacuum (UHV) environment (base pressure of 1 * 10 −10 mbar and under room temperature conditions. It is set up in an electromagnetically shielded box and is situated on a heavy concrete block which is decoupled from the building to minimise vibrations 22 , during the measurement, only ion getter pumps are used to pump the chamber. The tungsten tips are prepared by electrochemical etching in a 20 wt% NaOH solution and subsequent self-sputtering and annealing procedures in UHV. The measurements are conducted in constant current mode, with typical currents in the range of 10 nA, yielding electron doses in the range of 10 5 electrons per second per Å 2 . Both the illumination area and the emission voltage are usually stable over the course of a measurement session, i.e. over a few hours.
To create an undistorted reference wave, which is vital for LEEH imaging, an atomically clean, electrontransparent substrate is required. SLG fulfils these requirements as it is transparent to electrons in the relevant energy range and can be prepared in an ultraclean fashion 26 . Furthermore, graphene is conductive, which reduces charge-induced distortions and artefacts. In particular, a strong overlap of the electronic states of the graphene with those of the proteins indicate a critical role of the graphene in avoiding molecular charging during the electron irradiation 27 . Additionally, its weak interaction with biomolecules minimizes structural changes to the imaged proteins resulting from substrate interactions 28 .
For a detailed description of the experimental setup, the tip preparation and characterization as well as the graphene and protein sample preparation see Szilagyi 22 .
The data presented in this paper has been collected over the course of several experiments for both holo-and apo-transferrin. While not all data has been acquired using the same tip, the tip parameters have been kept as similar as possible to ensure high-quality hologram data. Each hologram is reconstructed at a range of tip-sample distances resulting in a z-stack of two-dimensional images. The focal distance is determined from this stack via the sharpness of the images. Because of the geometrical nature of the magnification, the determination of the focal distance also directly yields the size of the field of view for each reconstructed image. As this is done separately for each image, reconstructions obtained from holograms acquired with different tips or different imaging parameters can directly be compared.

Sample preparation.
To achieve the ultraclean sample conditions required for LEEH imaging, the proteins are deposited on SLG by native ES-IBD 17 . This not only allows a clean and controlled sample preparation, but also ascertains that the deposited molecules are chemically intact via mass spectrometry and mass selection procedures before deposition 2,3,17,29 . The spray solutions were created from lyophilized apo-and holo-transferrin (Sigma Aldrich) dissolved in 200 mM ammonium acetate and purified via buffer exchange columns. Nanoflow pulled glass capillary tips, operated at low voltages in the range of 1-1.5 kV were employed to retain a native state of the proteins during the electrospray process. The concentration of the spray solutions was in the range of 0.5-1 mg/ml. The proteins are soft-landed (kinetic energy upon landing below 5 eV per charge) on SLG in UHV. The coverage is controlled by monitoring the deposited charge, using a picoampmeter, to ensure sparse samples that allow the imaging of individual molecules. Native mass spectrometry. Native mass spectra ( Supplementary Fig. 1) were recorded on a Q Exactive UHMR Orbitrap mass spectrometer (Thermo Fisher Scientific). Protein solutions in 200 mM ammonium acetate were prepared and sprayed as described above. General instrument conditions were as follows: Source DC